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PubMed1995

Flagellar structure in normal human spermatozoa and in spermatozoa that lack dynein arms.

Human sperm flagella were analyzed by electron microscopy and computer averaging in order to characterize normal flagella and to detect differences between normal and mutated spermatozoa. The A-tubules of normal spermatozoa were seen to have 13 protofilaments and a lumen containing a 'pentagon' and a 'sickle'. The incomplete B-tubule was seen to have 10 protofilaments with an angular separation such that a complete circle would have 16 protofilaments. A thin '11th filament' is located at the inner border between A- and B-tubules and, in the centriole, also between B- and C-tubules. The tail end piece has 18 microtubules of a conventional appearance. We conclude that the 9 axonemal doublets split distally into 2 microtubules and that normal microtubules with 13 protofilaments can grow from the incomplete B-tubules. The cell membrane in the end piece has a glycocalyx with regular periodicity. Spermatozoa from a man suffering from the immotile-cilia syndrome was also analyzed. His sperm flagella were seen to be abnormal in that the dynein arms are lacking, and, that the sickle is incomplete. In other respects his immotile spermatozoa were normal; spokes and central projections have the same appearance as in normal spermatozoa.

Dyneins↗

PubMed1993

Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime.

While washing of human sperm cells by centrifugation and resuspension is a procedure in widespread use, there have been indications that this procedure per se may be harmful to the cells. The objective of this study was to investigate this question. To this end, a method for the clean separation of motile human spermatozoa from seminal plasma in the absence of centrifugation was developed, using a modified swim-up procedure, in which liquefied semen was mixed with an equal volume of 30 mg/ml dextran in medium, and the mixture overlaid with medium containing 5 mg/ml bovine serum albumin, forming two discreet layers with stable interface. The percentage of motile cells in a given sample was consistently > 80% immediately after recovery. Damage to the cells was assessed by loss of motile cells during incubation up to 96 h post-recovery. Comparison of aliquots of spermatozoa obtained by the dextran swim-up procedure showed that the aliquot subjected to centrifugation had 4 +/- 3% motile cells after 48 h, while the untreated aliquot had 52 +/- 12%. The aliquots showed no difference 1 h post-recovery. Similar results were obtained with spermatozoa that had been centrifuged in seminal plasma and resuspended in fresh plasma, then recovered by dextran swim-up. The delayed onset of motility loss in the centrifuged samples implies that this treatment induces sublethal damage in the cells. Comparison of the standard swim-up and Percoll gradient methods for sperm recovery, both of which involve centrifugation steps, showed decline in motility of the samples similar to that seen with dextran swim-up of centrifuged cells. We conclude that centrifugation per se induces sublethal damage in human spermatozoa, independently of treatment method, and suggest that recovery methods for human spermatozoa which avoid centrifugation might partially alleviate the damage incurred by these cells during cryopreservation.

Adult↗

PubMed1988

Heavy metals and human spermatozoa. III. The toxicity of copper ions for spermatozoa.

The dissolution of copper ions from copper metal into a saline medium in vitro was quantified using a colourimetric assay. The presence of spermatozoa enhanced this dissolution and increasing the protein content of the medium further increased the rate of dissolution. Approximately 17% of the copper released was either tightly bound to the spermatozoa or was within the cell and could not be removed by repeated washing. Once spermatozoa were immobilized, they could not be revived by washing and repeated changes of medium, by addition of copper specific-chelating agent or by extensive dialysis. When the toxicity to spermatozoa of cuprous and cupric ions was compared with copper metal, it could be shown that the quantity of cupric ions required (0.2-0.4 mg/ml) was in excess of the total quantity of copper released into solution. The quantity of cuprous ion required (0.08-0.16 mg/ml) to exert similar toxic effects to copper, was within the range of copper released from the metal. Under the conditions of this study, it is possible that cuprous ion would be oxidised to the cupric form generating free radicals in the process. It is not known whether the toxic effect is due to the cuprous ion, per se, or to radicals generated in its oxidation. Increasing the protein content of the medium to levels similar to low (8 mg/ml) and high (64 mg/ml) values reported in human uterine fluid increased the dissolution rate of copper but also offered some protection against the toxic effects of copper metal and cuprous and cupric ions.

Copper↗

PubMed2006

A heparin-binding membrane protein from goat spermatozoa immobilizes spermatozoa in the presence of complement.

OBJECTIVE: To characterize the major membrane protein of goat spermatozoa. DESIGN: Basic research. SETTING: Samples collected from local slaughterhouse and study conducted in an academic research environment. PATIENT(S): Goat epididymal tissue. INTERVENTION(S): Goat epididymal tissues were collected immediately after slaughter and the spermatozoa were isolated within 2 hours. Sperm immobilization test was performed with motile spermatozoa at 32 degrees C within 3-4 hours of collection and isolation of the cells. MAIN OUTCOME MEASURE(S): The heparin-binding sperm membrane protein (HBSM) of goat is insensitive to trypsin and its antisera immobilize spermatozoa in presence of complement. RESULT(S): Forty-two percent of membrane protein could be extracted with 0.25% (wt/vol) 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) from the isolated sperm membrane. By heparin-affinity chromatography, 46% of the extracted protein was recovered. Positive hybridization with radiolabeled heparin on western transfer confirmed the heparin-binding property of the protein (HBSM). Heparin binding to HBSM is an ionic bondage and can be disrupted by 1 M NaCl, as revealed by 86% recovery of the radiolabeled heparin in trichloroacetic acid-precipitated supernatant of [(125)I] heparin-HBSM conjugate. Heparin-binding sperm membrane protein is localized at the anterior region of the spermatozoal head. No detectable proteolytic fragment of HBSM was detected after limited digestion by trypsin. Heparin-binding sperm membrane protein antisera (1:10,000 titer) developed from rabbit did not recognize the denatured protein. The antisera inhibited spermatozoal motility in a complement-dependent manner. CONCLUSION(S): We suggest that the heparin-binding motif of the spermatozoal membrane protein might be required in modulation of the spermatozoal motility.

Animals↗

PubMed2006

Embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa in men with obstructive azoospermia.

OBJECTIVE: To determine whether the injection of testicular spermatozoa results in more viable embryos (higher implantation rate) than injection of epididymal spermatozoa in cases of obstructive azoospermia. DESIGN: Retrospective analysis of 265 cases of testicular sperm aspiration (TESA) and percutaneous sperm aspiration (PESA), including 185 cases of obstructive azoospermia. SETTING: Private Infertility clinic. PATIENT(S): None, charts review. INTERVENTION(S): None, charts review. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (PR), implantation rate. RESULT(S): Although fertilization rates were higher in the PESA group, implantation rates were significantly better in the TESA group. There was also a trend to higher ongoing PR and lower miscarriage rates in TESA cases. CONCLUSION(S): In cases of obstructive azoospermia, embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa.

Brazil↗

PubMed1982

Use of boar spermatozoa for artificial insemination. II. Fertilizing capacity of fresh and frozen spermatozoa in gilts inseminated either at a fixed time or according to Walsmeta readings.

A fertility trial was conducted comparing two methods of defining the optimum time to inseminate frozen boar semen. One hundred sixty-four gilts were inseminated, one-half at a fixed insemination time of 32 to 34 h after calculated onset of estrus and the remainder were inseminated on the basis of changes in the vaginal mucosal electrical resistance. Inseminations were made when the instrument (an inversely scaled ohmmeter) reading fell between 54 to 64 on a scale of 1 to 100. Both fresh and frozen boar semen were used. Pregnancy rates and live embryos/gilts from inseminations made at the fixed time and inseminations made on the basis of ohmmeter readings did not differ significantly for either fresh of frozen-thawed spermatozoa. Pregnancy rates and number of live embryos/gilt were significantly higher for gilts inseminated with fresh spermatozoa than for those inseminated with frozen-thawed spermatozoa (82%, 11.6 vs 61%, 8.2, respectively). Inseminations were conducted from January to September. Pregnancy rates were significantly higher for inseminations made for the first quarter or the year (January, February and March) than for inseminations made for the third quarter (July, August and September). No benefit was derived from using an ohmmeter to time insemination with frozen-thawed boar spermatozoa.

Animals↗

PubMed2003

Different fertilization rates between immotile testicular spermatozoa and immotile ejaculated spermatozoa for ICSI in men with Kartagener's syndrome: case reports.

We report two cases of infertility treatment in couples where males suffered from Kartagener's syndrome (KS) and a total absence of motile sperm in the ejaculate. A total of three ICSI cycles was carried out. In all cycles, viable ejaculated or testicular spermatozoa were selected using the hypo-osmotic swelling (HOS) test. Case 1: In the first ICSI cycle total fertilization failure occurred after using ejaculated spermatozoa. In the following cycle testicular spermatozoa were used for ICSI, resulting in 75% fertilized oocytes and a pregnancy. Case 2: In the same ICSI cycle 50% of the oocytes were injected with ejaculated and 50% with testicular spermatozoa. The fertilization rates were 44 and 56% respectively and high quality embryos were achieved in both groups. One single embryo derived from testicular sperm was transferred with a resulting singleton pregnancy. In conclusion, testicular sperm for ICSI seem to have reliable fertilization capacity in men with KS, while ejaculated sperm, even if tested viable, seem more unpredictable. HOS test for selection of viable sperm for ICSI is recommended when ejaculated as well as testicular sperm are used for ICSI.

Adult↗

PubMed2003

Sperm tail flexibility test: a simple test for selecting viable spermatozoa for intracytoplasmic sperm injection from semen samples without motile spermatozoa.

PURPOSE: The objective was to describe the results of the injection of immotile spermatozoa with flexible tails when only immotile spermatozoa are present in the semen sample. METHODS: A retrospective study was conducted to analyze the procedure results for 10 couples who participated in our intracytoplasmic sperm injection program. The sperm tail was considered flexible when it moved up and down independently of the head movement, and it was considered inflexible when the movement occurred together (tail plus head). The fertilization and pregnancy rate were analyzed. RESULTS: The normal fertilization rate (presence of 2 pronuclei) was 30.3% (40/132), and the abnormal fertilization rate (presence of less than or more than 2 pronuclei) was 6.81% (9/132). A total of 52 embryos were obtained with 9 transfer procedures performed (pregnancy rate: 11.12%). CONCLUSIONS: The sperm tail flexibility test (STFT) is an easy and cost-effective way for selecting viable immotile spermatozoa and can be used as an alternative method for determining the viability of spermatozoa. This test seems to be a simple and risk-free method when compared to the swelling test.

Adult↗

PubMedDate not supplied

[Species specific motility patterns of hyperactivated mammalian spermatozoa and quantitative analysis of the hyperactivation of bull spermatozoa].

Movement tracks of spermatozoa of human, lion, tiger, cow, pig and sheep are recorded by dark field photography (fluid layer thickness 16.7 microns, exposure time 1 s). Comparison before and after capacitation by an incubation of 2 h in modified tissue culture medium TCM 199 with 10% fetal calf serum resulted in two quite different patterns of hyperactivated spermatozoa: 1) Tracks are broadened due to enlarged lateral head displacement or radius of rotating head movements respectively (tiger, lion) and show beside that a markedly increase in erratic motility (human). 2) In the studied species of artiodactyla, cow, pig and sheep, a qualitative new, panicle-like pattern arised as a result of superposition of spermatozoa head pendular movements around the axis of forward motility and the other one around the head axis. This new type of tracks allows a simple quantitative analysis of hyperactivation of bull spermatozoa, first described in this report. The method is applied to investigations on efficacy of capacitation media and provides evidence for high individual differences of semen donors in capacitation success.

Animals↗

PubMed1990

HLA-D determinants are expressed on human seminal cells other than spermatozoa but not on purified spermatozoa.

Purified human spermatozoa do not regularly stimulate lymphocytes in spermlymphocyte cultures (SLC). This lack of consistent stimulation was found not to be dependent on sperm/lymphocyte ratio or on culture peak response time. A few weak stimulations obtained are not HLA-D or sex dependent. Contaminating seminal cells other than spermatozoa (SC non-SZ) may be responsible for the high stimulations found in SLC by others, since, in our hands, purified suspensions of SC non-SZ stimulate allogeneic lymphocytes in an HLA-D-dependent fashion, and such responses are abrogated by anti-HLA-DR monoclonal antibodies. These functional data confirm our previous finding of HLA-DR molecules on SC non-SZ by absorptions (and a lack of expression on spermatozoa) and suggest the concomitant expression of HLA-DQ and -DP products.

Antibodies, Monoclonal↗

PubMedDate not supplied

Relation between spermatozoa motility and pregnancies obtained during a twenty-year follow-up period spermatozoa motility and fertility.

The clinical fertility of 1077 men with an infertility problem investigated with semen analysis including assessment of spermatozoa motility during the years 1950-52 was studied 20 years later using a questionnaire, replied by 785 (72.9%). Spermatozoa motility was assessed with a subjective method with four degrees--excellent, good, impaired and poor. There were significant correlations between better motility and number of living children (p less than 0.01) and to shortest time--interval to first pregnancy (p less than 0.001). Motility was not correlated to abortions and otherwise pathological pregnancies. With a poor motility there was still a fairly good chance of obtaining living children (32.7%) compared with 58.7% for an excellent motility), and it is therefore concluded, that assessment of spermatozoa motility is only suited for classification of slightly and modestly reduced male fertility, and not for classification of severely impaired fertility.

Abortion, Spontaneous↗

PubMed1984

Nuclear androgen binding sites in the male rat. III. Late spermatids and spermatozoa in the testis, with an introduction to epididymal spermatozoa.

Nuclear androgen binding sites were examined in late spermatids (stages 12-19) which resisted sonication of homogenized testes of mature male rats. The measurement of unoccupied binding sites in salt extract of purified spermatid heads by nuclear exchange at -10 degrees C was developed and validated. As in the prostate, unoccupied nuclear androgen binding sites in sonicated testes were in low concentration, were not artefactual, and could be occupied both in vivo and in vitro by exogenous androgens, and uniquely in hemicastrated rats by endogenously compensated androgens in the remaining testis. The properties of occupied binding sites in salt extract of purified spermatid heads (measured by nuclear exchange at 4 degrees C for 48 or more hours with 5 nM [3H]dihydrotestosterone) were almost identical to those of occupied binding sites in nuclei of the ventral prostate, except for their concentration. However, levels of specific binding activity approaching 50 fmol/mg DNA could be expected in salt extract of spermatid pellets, by use of a sulfhydryl reducing agent (dithiothreitol) prior to salt extraction, a protease inhibitor (phenylmethylsulfonyl fluoride) in all buffers, and optimization of the sonication protocol. Nuclear androgen binding sites of sonicated epididymal spermatozoa, collected by retrograde perfusion of the cauda epididymidis, were found to be completely salt-resistant. These binding proteins could be extracted by 0.4 M KCl if dithiothreitol and dihydrotestosterone were incorporated into the sonication buffer, if phenylmethylsulfonyl fluoride was added to all buffers, and if the purified epididymal sperm pellet was treated with sarkosyl, a non-ionic detergent, just before salt extraction. The salt extract of epididymal spermatozoa which were treated as described above contained two binding components: a soluble form which was eluted from hydroxylapatite by increasing concentrations of phosphate buffers, and a non-soluble form, free of DNA, which remained in the hydroxylapatite column, and which contained most of the androgen binding sites. Affinity (Kd) of dihydrotestosterone to the soluble and insoluble fractions of the steroid-binding protein complex was determined to be 0.7 and 0.1 nM, respectively. Salt-resistance of binding proteins in germ cells was shown to develop significantly in the last stages of spermiogenesis.

Animals↗

PubMed2005

Effects of glutamine on post-thaw motility of stallion spermatozoa: an approach of the mechanism of action at spermatozoa level.

The cryoprotective effect of l-glutamine and an approach of its mechanism of action, in preserving motility of stallion spermatozoa during the freezing-thawing process, were studied. In Experiment 1, thirty-six ejaculates were collected from six stallions (two good, two middle, and two of poor sperm freezability) and were diluted with 10 different freezing media derived from INRA 82 medium supplemented with 20 mM HEPES and 2% (v/v) centrifuged egg yolk (BM). After thawing, sperm motility was evaluated by a computer-assisted semen motility analyser. The effects of glutamine and glycerol at different concentrations on post-thaw sperm motility were studied. A possible interaction between medium and semen freezability was investigated. Only the 50 mM glutamine + 2.5% glycerol medium significantly improved sperm motility compared to classical freezing medium (2.5% glycerol). The presence of glutamine at 50 mM was not sufficient to offset the need to use glycerol in the freezing extender. The use of glutamine at a higher concentration >100 mM in the presence of 2.5% of glycerol was toxic. Reducing the glycerol proportion from 2.5% to 2 or 1.5% in the presence of glutamine at 50, 75, and 100 mM had no influence on post-thaw motility of semen of middle and good freezability. Moreover, the substitution of 2.5% glycerol by 50 mM glutamine in BM, did not significantly change the post-thaw motility of semen of good freezability. In Experiment 2, 3H-glutamine and 3H-glycerol were used to study the kinetics of penetration of glutamine and glycerol in sperm cells. The radioactivity of each radio-labelled semen pellet was measured at different times (0, 15, 30, 60, 90, 120 min), by using a Packard tri-carb 4530 apparatus. The percentages of incorporated radioactivity (%IRA) in semen pellets were calculated at different times. The %IRA of 3H-glycerol in semen pellets were significantly higher than the %IRA of 3H-glutamine. The %IRA of 3H-glycerol in semen pellets increased greatly from time 0 to 60 min, and then it is stabilized from 60 to 120 min. In contrast, the %IRA of 3H-glutamine in semen pellets increased slightly from 0 to 60 min, then it stabilized until 120 min. These experiments demonstrate that glutamine has a synergistic cryoprotective effect with glycerol on cryopreservation of stallion spermatozoa, and suggest that glutamine acts at the extra-cellular level, independently of glycerol.

Animals↗

PubMed1992

Density differences between spermatozoa with antisperm autoantibodies and spermatozoa covered with antisperm antibodies from serum.

Autoantibody-carrying spermatozoa from infertile men were processed using a discontinuous Percoll gradient. The treatment yielded a sperm fraction with significantly reduced antibody loading on the sperm head but did not affect antibody binding to the sperm tail. The immunodepleted sperm preparation demonstrated fertilizing ability when used in in-vitro fertilization. Normal, antibody-free semen samples were coated in vitro with antisperm antibodies from serum, followed by Percoll gradient centrifugation. No significant separation of antibody-free spermatozoa was obtained, regardless of the antibody binding site.

Antibodies↗

PubMedDate not supplied

Monosaccharide transport across membranes of human spermatozoa. I. Development of a radiochemical method of measuring monosaccharide uptake by spermatozoa.

A method for determination of the monosaccharide uptake into human spermatozoa is described. Intracellular monosaccharide concentrations were calculated on the basis of determination of intracellular radioactivity after incubation of the cells with labelled monosaccharide and using a mathematical procedure to approximate the intracellular space of spermatozoa. The D-fructose uptake depends on the extracellular D-fructose concentration in a hyperbolic manner. Half maximal saturation is present at 4,1 mM. This corresponds closely with the lowest limit of D-fructose concentration in human fertile semen.

Biological Transport↗

PubMed1976

[Round-headed spermatozoa: a rare andrologic finding ("globe-headed spermatozoa", "globozoospermia")].

The case of an infertile patient is reported who had 100% round-headed ("globe-headed") spermatozoa without acrosomes in his ejaculate. The spermatozoa were studied by light and phase contrast microscopy, as well as by transmission and--for the first time--by scanning electron microscopy. Determination of acrosin showed no enzyme activity. We propose the term "globozoospermia" for this condition which was first described in 1971. It is discussed whether it may be considered as a symptom or a distinct disease.

Acrosin↗

PubMed1977

[Achievements in the study of the development and ultrastructure of spermatozoa (on the 300th anniversary of the discovery of spermatozoa)].

A review of principal advances in studying spermiogenesis and fine structure of spermatozoa is given on the occasion of the 3rd centennary of the discovery of spermatozoa by Loevenhuk. The most important achievements are the discovery of acrosome and its role in fertilization, the discovery of centriole and its role in the formation of flagellum, the discovery of axial complex and the role of its components in the flagellum movement. A study of spermatogenesis has revealed the syncytial nature of spermatogenic epithelium, synaptinemal complexes and their role in chromosome conjugation. The role of nuclear membrane in chromosome conjugation was established, the ultrastructural and biochemical transformation of chromatin was investigated, as well as that of microtubules in spermatogenesis. Some other discoveries were made which are of great importance for diagnosis of sterility.

Acrosome↗

PubMed1992

A comparison between in vitro fertilization and microinjection of immobilized spermatozoa from bulls producing spermatozoa with defects.

The objectives of this study were to compare the fertilization rate of bovine in vitro matured oocytes by in vitro fertilization (IVF) and by microinjection of a single spermatozoon (MI) and to relate these rates with fertility reported for these bulls in artificial breeding. Bull A (Holstein) had a nonreturn rate of 75%. Semen from this bull is routinely used in our standard IVF procedure. Bull B (Ayrshire), used regularly in artificial breeding and related to bull D, had a nonreturn rate of 69.2%. Bull C (Brown Swiss), with a chromosomal translocation and trisomy, achieved a nonreturn rate of 42%. Bull D (Ayrshire) produced nonmotile spermatozoa (SPZ) and had an abnormality described as "tail stump defect." No pregnancies sired by bull D have been reported. Oocytes were either fertilized in vitro by capacitated SPZ or by microinjection of a single immobilized SPZ into the ooplasm. SPZ were treated with 0.1 microM A23187 and used for IVF. For microinjection SPZ were cocultured for 5 h with bovine oviduct epithelial cells (BOEC) and then immobilized by freezing and thawing twice without cryoprotectant. A single batch of killed SPZ (stored at -25 degrees C) was used for all microinjections. All oocytes were cultured in Medium 199 for 22 h at 39 degrees C and subsequently fixed, stained, and examined for evidence of fertilization (i.e., female and male pronucleus formation, SPZ decondensation). Fertilization rates following IVF with semen from bulls A, B, C, and D were 80%, 54%, 1%, and 2%, and following microinjection were 39%, 22%, 21%, and 34%, respectively.

Animals↗